Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification

Abstract Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A‐B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A‐B55. We use specific inhibition of PP1 and PP2A‐B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A‐B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.

Thank you again for submitting your work to Molecular Systems Biology.We have now heard back from the three reviewers who agreed to evaluate your study.As you will see below, the reviewers think that the study is a relevant contribution to the phosphatase field.They raise however a series of concerns, which we would ask you to address in a revision.I think that the reviewers' recommendations are rather clear so I do not see the need to repeat any of the points listed below.All issues raised by the reviewers need to be satisfactorily addressed.As you may already know, our editorial policy allows in principle a single round of major revision, so it is essential to provide responses to the reviewers' comments that are as complete as possible.I understand that the required revisions are substantive.Please feel free to contact me in case you would like to discuss in further detail any of the issues raised or if you would like to share your revision plan with me.I would be happy to schedule a call.
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Overall, this is a large body of work that makes significant contributions to our understanding of phosphoprotein phosphatase biochemistry and signaling.As currently presented, however, the second half of the manuscript, focused on the RVxF interactions, is hard to follow.In particular, the descriptions of the analysis describing how RVxF motif binding overrides catalytic domain preferences are not clear.Several major and minor revisions (largely non-experimental) are suggested in order to improve the depth of the analysis and also the clarity of the manuscript.Given the scope and significance of this study, both in terms of methods and concepts, a refined version of this manuscript would be a very welcome contribution to the phosphatase signaling field.
Suggested major points to address: 1. Can the authors clarify how many beads they are imaging for each pooled dephosphorylation reaction?Are there multiple beads per peptide, and if so, how is this treated in the data analysis?Related to this, does this depth matter?In other words, if you have many beads in one reaction corresponding to the same peptide, and the rates for these beads are averaged, does this improve the method?This would be distinct from doing multiple separate dephosphorylation reactions (almost like technical vs "biological" replicates).2. In Figure 1F, it's very difficult to evaluate how well the dephos score correlates with data from the orthogonal assay, as currently plotted.It might be helpful to either plot a scatterplot of the comparison and/or report a correlation coefficient between the datasets.Since the rank-order of substrates via dephos score and phosphate release score don't appear to be identical for the panel tested (although they are close), can the authors discuss the potential sources of these discrepancies? 3. Can the authors explain how they know that Nipp1 and thio-Arpp19 are selective inhibitors of PP1 and PP2A-B55, respectively, and not of other PPPs?Is it possible to do an IP or other experiment that might show if the two inhibitors are binding to other phosphatases in the cell lysates, or perhaps such an experiment has been published previously?This is particularly important, given the very low overlap between the PP1 dataset in this paper and that from references 10 and 11. 4. It is surprising that, in panel 2D, those sequences that are biased toward PP1 over PP2A-B55 appear to have a +1 proline, but that a +1 Pro is a preferred residue for PP2A-B55 and a disfavored residue for PP1 in the proteomics experiments (Figure 2E).Can the authors explain/discuss this discrepancy more?It is already suggested in the text that this might be due to prolyl isomerization, or do other cellular interactors driving co-localization and overriding the anti-proline preference observed in vitro.Another possibility is that the favoring of a proline at the +1 position is dependent on some other sequence feature that is not present in the MRBLE library but is present in the subset of the proteome that is enriched.This should be examined.Yet another possibility is that PP2A-B55 is not being selectively inhibited in the proteomics experiment, and the observed +1 P signal is coming from another phosphatase.Finally, as hinted at by the authors, it may simply be the case that the cellular substrates of PP2A-B55 are not enzymatically optimal, because that might not be what is optimal for cell signaling (i.e. a faster dephosphorylation reaction might not be ideal for what the cell needs in order to function properly).Thus, other recognition features of the substrates are sufficient to select the substrates, and the +1 P keeps the rate of dephosphorylation low.Overall, these points need further discussion.5. Given the issues posed in the previous point, it would be worthwhile to examine INCENP phosphorylation by PP2A-B55 at the peptide level with a mutation at P60 (perhaps P60A).Cellular assays with this mutant would also be interesting.6.For Figure 3C, the S61E mutation is tested in cells, and it is suggested that Cdk1 can still phosphorylate this mutant.The figure legend indicates that the data shown are one representative dataset from three replicates.For the quantification, data from all three replicates, along with error bars, should be shown.
7. For all of the proteomics analysis, the authors should include a full description of the statistical analysis used to identify significant phosphosites or interactors (specifying number of replicates, fold-change cutoffs, and p-value cutoffs).Related to this, it would be helpful to see volcano plots of all of the data, where relevant.And the full volcano plots corresponding to the data in figure 4A,B should be shown in the SI. 8.For the analysis in Figure 4D, the authors are asserting that phosphosites in Ki67 near the RVxF motif more frequently have the SP motif than the TP motif, whether regulated by PP1 or not.By contrast, sites away from Ki67 that are PP1-regulated more frequently have a TP motif instead of SP motif.What is unclear from this analysis, and from the figure, is whether this bias actually reflects an effect of RVxF-mediated localization vs. intrinsic catalytic domain preferences, as suggested, or if this can be explained by the frequency of TP vs SP phosphosites in the protein.What might help make this distinction is to know how many sequences were used to make up each logo.9. Related to the previous point, a similar analysis could be done for the data in Figures S7 and S8 -The sequence preferences for PP1-and PP2A-regulated sites across the proteome (with and without an associated RVxF site) should be normalized to the all phosphosites on RVxF-associated vs not-associated sites.This would really tell illustrate whether presence of the RVxF motif impacts the substrate sequence tolerance of PP1-and PP2A.In other words, is the dephosphorylation of pSer residues by PP1 actually due to the presence of an RVxF motif nearby, or is it the case that phosphorylation sites near RVxF tend to be pSer sites rather than pThr sites?Same goes for net charge around the phosphosite.What is the distribution of net charge around phosphosites near RVxF vs away from RVxF sites?10.Were any peptides in the RVxF MRBLE experiment also characterized by ITC or any other classical titration binding experiment?It would be helpful to see how well the Kd values from two orthogonal methods correlate.11.It is unclear Suggested minor revisions: 1. Very minor point: In the intro, paragraph 2, it might be useful to clarify whether PP1 and PP2A are the primary PPPs in just eukaryotic cells, or also organismal clades.The current text just says that they are responsible for the majority of Ser/Thr dephosphorylation in cells.2. For the sequences in Figure 1D, it appears that the phospho-Ser and phospho-Thr residues are labeled as Sp or Tp, whereas these phospho-residues are most conventionally written as pS/pT, which makes reading the sequences a bit confusing.3. It would be helpful if the logos in figure 2E were in the same color scheme as the logos in the rest of Figure 2. 4. Similar to the previous point, the coloring of the sequences in Figure 2D is the opposite of what is in the logos: in the logos (as is often conventional), D/E are red, and K/R are blue, but in the listed sequences the opposite color scheme is used.5.This may be a file processing issue, but Figure S7 is too blurry to read the labels on the nodes in the network diagram.6.In general, it is unclear what the reader stands to learn from the two interaction network diagrams.Related to this, it is quite difficult to discern where PP1 and PP2A-B55 are in these maps.Perhaps the authors could highlight/explain parts of the interaction maps that would be of significance/value to the reader?7.For non-specialists, it may be helpful to define INCENP and explain its role in mitosis.8. Minor question: is "dephosphorylome" a common term?If not, while this is a purely stylistic consideration, it may by simpler for the readers if some more common phrasing is used.9.In the last sentence of the first paragraph, there is a typo: "phosphoyraltion" Reviewer #2: In this study, Hein and co-workers address the complex topic of cellular protein phosphatase selectivity.To do this they use mass spectrometry approaches to analyse dephosphorylation of peptide libraries in vitro by PP1 and PP2A-B55, coupled with global identification of cellular substrates for these enzymes as an in vivo correlate.In summary, there is a considerable amount of high-quality work in the study, and the new data will provide a valuable additional resource for the field.

Specific points:
In Figure 3, INCENP is taken as an example to test a key feature of the dephosphorylation motif.Previous work has shown that INCENP relocalisation from pericentromeric chromatin to the anaphase spindle requires dephosphorylation at T59, and can be prevented with a T59E mutant.This forms a switch controlling binding to a motor protein KIF20A at the onset of anaphase.The charge introduced in the S61E mutant may directly prevent binding to KIF20A.Additional controls are necessary for Figure 3D-E, where a T59A/S61E mutant should be tested.If the authors are correct, then this should to the anaphase spindle since it cannot be phosphorylated.If it fails to localise then defective anaphase localisation is due to a blockade of KIF20A binding and not a dephosphorylation defect.
The authors note that while the overlap between PP2A-B55 substrates in vivo in their data and previous work is good, the overlap for PP1 is less clear.There are good reasons for this which need a little more explanation.This also highlights some caveats to the peptide approach, which is important since PP1 is generally thought to act in complex with other regulatory subunits that modify substrate selection.Using PP1 alone in the in vitro approach, and taking Nipp1 as the sole method to modulate PP1 in vivo may not address the full range of PP1 substrates.PP2A-B55 is probably only regulated by Arpp19/ENSA, so the approach would be more general in terms of the range of substrates.
Figure 4 is confusing and it is difficult to extract a clear message.Is there some other way to represent the choice of network diagram is not obvious to me, since the study isn't focussed on the interactions between proteins, but rather the sequences of individual phosphatase sites.Is there some way to strengthen the link to Figure 5 in terms of testing whether dephosphorylation alters behaviour of a key PP1 regulated protein or proteins.In that sense the data in Figure S10 is important and could be moved to a main figure.

Reviewer #3:
This manuscript describes an adaptation of the MRBLE peptide:protein interaction assay to a format (MRLBE-Dephos) that can be used to measure time-dependent peptide dephosphorylation by phosphatases.The authors have subsequently used this assay to compare the substrate selectivity of two key phosphatases (PP1 and PP2A-B55), either purified or as present in mitotic cell lysates.The MRLBE-Dephos assay is novel and generated interesting data, but needs to be better validated.Also, while the observed determinants of substrate selectivity of PP1 and PP2A-B55 are largely in agreement with previously established determinants, as derived from other experimental approaches, there are obvious differences that need to be further explored.My detailed comments are detailed below.

Specific comments
1. What are the concentrations of the peptides and phosphatases during the MRLBR-dephos assay?The method section only specifies volumes or amounts.This is essential information needed for for comparison with other phosphatase assays.
2. The MRLBE-Dephos assay measures remaining phosphate (high signal less high signal), which is less sensitive than classical assays that quantify phosphate release (no signal signal).This should be commented upon.
3. In all MRLBE dephosphorylation assays (Fig. S1), the dephosphorylation apparently stops before completion.The reason for this is not explained.Also, since the dephosphorylation is not linear with time (Fig. S1), it is not clear how the dephosphorylation score can be accurately derived from the first and last time points.4. A comparison of classical phosphatase assays (phosphate release) and the MRLBE-Dephos score (Fig. 1E) shows essentially no dephosphorylation for some peptides with the first assay but still very significant dephosphorylation with the second assay.This indicates that the MRLBE-Dephos assay not only measures phosphatase activity but, e.g., also loss of bead-associated peptide.
5. Since the peptides were synthesized on the beads, it should be explored whether binding to the beads affects their properties as substrates.
6.It is biologically not relevant to compare the substrate selectivity of the PP1 monomer, which does not exist in vivo, with a PP2A-B55 holoenzyme.Also, bacterially expressed PP1 has a substrate selectivity that is very different from that of native PP1 (https://doi.org/10.1111/j.1432-1033.1993),making a direct comparison even more irrelevant.
7. The most striking feature of the comparison between purified PP1 and PP2A-B55 is their similar activity on a broad range of peptide substrates (Fig. 1D; Fig. 2A-D).It is difficult to grasp how the observed minor differences can explain the clearly distinct, albeit somewhat overlapping functions, of these phosphatases in vivo.The differences in substrate specificity would most likely have been much more obvious if a specific PP1 holoenzyme and PP2A holoenzyme were compared, with the free catalytic subunits as reference points (see previous comment).8.It is not correct to refer to 'benchmark in vitro data with in vivo substrates' (p8, line 1) when the actual comparison is purified phosphatases versus phosphatases present in mitotic lysates.9.While thio-phosphorylated proteins are somewhat resistant to dephosphorylation, they will eventually also be dephosphorylated.Hence, the authors should examine to which extent thiophosphorylated Arpp19 is dephosphorylated in the adopted timeframe(s).Nipp1 is indeed a highly selective inhibitor of PP1 but it only prevents the dephosphorylation of a subset of substrates.This limitation should be indicated.10.Less than 10% of the identified mitotic phosphorylation sites (˃ 38,000) were affected by PP1 or PP2A-B55 inhibition.This indicates that the inhibition of these phosphatases was incomplete and/or that it is incorrect to that these phosphatases represent the major mitotic-exit phosphatases.11. p9, first line: H3S29 does not exist.Did the authors aim to refer to H3S28? 12.The limited overlap between PP1 substrates in mitotic lysates and previous substrate-mapping studies is worrysome, and is further evidence that PP1 was only partially blocked in the mitotic lysates.
13.The identified PP1 and B55 interactors (Table S2) are poorly described and not independently validated.Are they regulatory subunits and/or substrates?How much overlap is there with known interactors?14.The interaction of PP1 with RVxF motifs and its regulation by mitotic phosphorylation of residues within or next to the RVxF motif has already been studied in some detail by various research groups and is not properly referred to in the manuscript.

Reviewer #1:
This manuscript Hein et al. presents a very exciting high-throughput method to characterize the substrate sequence specificities of protein phosphatases: MRBLE-Dephos.This method builds on the MRBLE platform developed by the Fordyce lab with the added detection of phosphopeptides dephosphorylation as a function of time.The method allows for rapid, quantitative comparison of dephosphorylation rates for 94 peptides at a time, and this was applied with a tailored peptide library to analyze pT/pS-proximal sequence preferences for the phosphatases PP1 and PP2A-B55.Juxtaposition of the sequence preferences identified using MRBLE-Dephos with proteomics data from this study and published studies allow for a clear assessment of the extent to which catalytic domain sequence specificity governs substrate recognition in a cellular context.The manuscript ends by examining the contributions of a secondary binding motif (RVxF) in PP1 substrate selection, by juxtaposing interactomics experiments, phosphoproteomics data, and MRBLE binding assays.
Overall, this is a large body of work that makes significant contributions to our understanding of phosphoprotein phosphatase biochemistry and signaling.As currently presented, however, the second half of the manuscript, focused on the RVxF interactions, is hard to follow.In particular, the descriptions of the analysis describing how RVxF motif binding overrides catalytic domain preferences are not clear.Several major and minor revisions (largely non-experimental) are suggested in order to improve the depth of the analysis and also the clarity of the manuscript.Given the scope and significance of this study, both in terms of methods and concepts, a refined version of this manuscript would be a very welcome contribution to the phosphatase signaling field.

Suggested major points to address:
1. Can the authors clarify how many beads they are imaging for each pooled dephosphorylation reaction?Are there multiple beads per peptide, and if so, how is this treated in the data analysis?Related to this, does this depth matter?In other words, if you have many beads in one reaction corresponding to the same peptide, and the rates for these beads are averaged, does this improve the method?This would be distinct from doing multiple separate dephosphorylation reactions (almost like technical vs "biological" replicates).

To increase clarity, we now include an SI figure showing the number of beads profiled
per code in each experiment (Fig. EV1), which we also reproduce here (Fig. R1).1F, it's very difficult to evaluate how well the dephos score correlates with data from the orthogonal assay, as currently plotted.It might be helpful to either plot a scatterplot of the comparison and/or report a correlation coefficient between the datasets.Since the rank-order of substrates via dephos score and phosphate release score don't appear to be identical for the panel tested (although they are close), can the authors discuss the potential sources of these discrepancies?

In response to the Reviewer's request, we now include a scatter plot directly comparing the MRBLE:dephos dephosphorylation score with the amount of phosphate released in orthogonal assays along with a linear regression to help visualize correlation. This plot is now included as Fig. 1F (reproduced here as Fig. R2 for convenience).
3. Can the authors explain how they know that Nipp1 and thio-Arpp19 are selective inhibitors of PP1 and PP2A-B55, respectively, and not of other PPPs?Is it possible to do an IP or other experiment that might show if the two inhibitors are binding to other phosphatases in the cell lysates, or perhaps such an experiment has been published previously?This is particularly important, given the very low overlap between the PP1 dataset in this paper and that from references 10 and 11.

We appreciate these comments from the reviewer. Both Nipp1 and thio-Arpp19 has been extensively characterized as specific inhibitors of PP1 and PP2A-B55 by other labs. Nipp1 binds PP1 through and RVxF motif while thio-Arpp19 blocks the active site of PP2A-B55. The previous work characterizing Nipp1 and Arpp19 are cited in the manuscript. In the revised manuscript we write on page 9:
"We used the central domain of Nipp1 and thiophosphorylated Arpp19 (thio-Arpp19) as specific natural inhibitors of .Nipp1 prevents binding of RVxF-containing proteins to PP1, in this way preventing a large subset of PP1 holoenzymes from forming and thus blocking PP1 dephosphorylation.In contrast, thio-Arpp19 blocks the active site of PP2A-B55, effectively blocking activity."4. It is surprising that, in panel 2D, those sequences that are biased toward PP1 over PP2A-B55 appear to have a +1 proline, but that a +1 Pro is a preferred residue for PP2A-B55 and a disfavored residue for PP1 in the proteomics experiments (Figure 2E).Can the authors explain/discuss this discrepancy more?It is already suggested in the text that this might be due to prolyl isomerization, or do other cellular interactors driving colocalization and overriding the anti-proline preference observed in vitro.Another possibility is that the favoring of a proline at the +1 position is dependent on some other sequence feature that is not present in the MRBLE library but is present in the subset of the proteome that is enriched.This should be examined.Yet another possibility is that PP2A-B55 is not being selectively inhibited in the proteomics experiment, and the observed +1 P signal is coming from another phosphatase.Finally, as hinted at by the authors, it may simply be the case that the cellular substrates of PP2A-B55 are not enzymatically optimal, because that might not be what is optimal for cell signaling (i.e. a faster dephosphorylation reaction might not be ideal for what the cell needs in order to function properly).Thus, other recognition features of the substrates are sufficient to select the substrates, and the +1 P keeps the rate of dephosphorylation low.Overall, these points need further discussion.obvious, the dephosphorylation of specific "charged" TP sites does occur but not at a higher rate than the overall pool of TP phosphorylation sites.

The dephosphorylation preference of these TP sites by PP1 is not obvious in Figure 2E due to the large number of proline-directed phosphorylation sites in mitosis. To determine the enrichment of sequence elements, background correction is used for icelogos. Here, the background consisted of the non-regulated sites from our phosphoprotemics (Figure 2E). While the lack of dephosphorylation of SP sites by PP1 is
We have discussed some of the aspects in the revised manuscript on page 10. 5. Given the issues posed in the previous point, it would be worthwhile to examine INCENP phosphorylation by PP2A-B55 at the peptide level with a mutation at P60 (perhaps P60A).Cellular assays with this mutant would also be interesting.

We have updated the figure and shown below for convenience.
7. For all of the proteomics analysis, the authors should include a full description of the statistical analysis used to identify significant phosphosites or interactors (specifying number of replicates, fold-change cutoffs, and pvalue cutoffs).Related to this, it would be helpful to see volcano plots of all of the data, where relevant.And the full volcano plots corresponding to the data in figure 4A,B should be shown in the SI.

We have provided this information and provided volcano plots of Fig. 4A and B in the main figure 4 of the revised manuscript. Shown below for convenience.
8. For the analysis in Figure 4D, the authors are asserting that phosphosites in Ki67 near the RVxF motif more frequently have the SP motif than the TP motif, whether regulated by PP1 or not.By contrast, sites away from Ki67 that are PP1-regulated more frequently have a TP motif instead of SP motif.What is unclear from this analysis, and from the figure, is whether this bias actually reflects an effect of RVxF-mediated localization vs. intrinsic catalytic domain preferences, as suggested, or if this can be explained by the frequency of TP vs SP phosphosites in the protein.What might help make this distinction is to know how many sequences were used to make up each logo.9. Related to the previous point, a similar analysis could be done for the data in Figures S7 and S8 -The sequence preferences for PP1-and PP2A-regulated sites across the proteome (with and without an associated RVxF site) should be normalized to the all phosphosites on RVxF-associated vs not-associated sites.This would really tell illustrate whether presence of the RVxF motif impacts the substrate sequence tolerance of PP1and PP2A.In other words, is the dephosphorylation of pSer residues by PP1 actually due to the presence of an RVxF motif nearby, or is it the case that phosphorylation sites near RVxF tend to be pSer sites rather than pThr sites?Same goes for net charge around the phosphosite.What is the distribution of net charge around phosphosites near RVxF vs away from RVxF sites?

Figure R8.
There is no significant difference in the net charge.However, negatively charged amino acids (D and E) seem to be better tolerated in the +1 and +4 positions of phosphorylation sites on RVxF-containing proteins compared to non-RVxF proteins.

This analysis supports the conclusion that RVxF motifs can modulate substrate sequence preferences. We have included a line on page 15 in the revised manuscript to summarize this.
10. Were any peptides in the RVxF MRBLE experiment also characterized by ITC or any other classical titration binding experiment?It would be helpful to see how well the Kd values from two orthogonal methods correlate.

It is unclear
Suggested minor revisions: 1. Very minor point: In the intro, paragraph 2, it might be useful to clarify whether PP1 and PP2A are the primary PPPs in just eukaryotic cells, or also organismal clades.The current text just says that they are responsible for the majority of Ser/Thr dephosphorylation in cells.
We have clarified this.
2. For the sequences in Figure 1D, it appears that the phospho-Ser and phospho-Thr residues are labeled as Sp or Tp, whereas these phospho-residues are most conventionally written as pS/pT, which makes reading the sequences a bit confusing.

We have edited all panels of all figures to follow this convention as suggested.
3. It would be helpful if the logos in figure 2E were in the same color scheme as the logos in the rest of Figure

Corrected.
4. Similar to the previous point, the coloring of the sequences in Figure 2D is the opposite of what is in the logos: in the logos (as is often conventional), D/E are red, and K/R are blue, but in the listed sequences the opposite color scheme is used.
We have edited the colors of these sequences to reflect the standard convention.
5. This may be a file processing issue, but Figure S7 is too blurry to read the labels on the nodes in the network diagram.
We have tried to improve quality.6.In general, it is unclear what the reader stands to learn from the two interaction network diagrams.Related to this, it is quite difficult to discern where PP1 and PP2A-B55 are in these maps.Perhaps the authors could highlight/explain parts of the interaction maps that would be of significance/value to the reader?

We aimed at providing these networks as a resource for the scientific community. We think these networks will be relevant for many researchers working on different biological pathways regulated by these phosphatases. We have now highlighted a few examples of information that can be extracted from these networks. We write on page 12:
Our networks revealed that both PP1 and PP2A-B55 dephosphorylate a diverse set of proteins regulating core biological processes involved in mitotic exit as well as interphase functions.Several known RVxF containing proteins such as KNL1, RIF1 and RepoMan (CDCA2) were substrates of PP1 as expected but our network analysis also suggested that these proteins coordinate dephosphorylation of associated proteins.As an example, KNL1 could scaffold PP1 dephosphorylation of CENPC S261, a phosphorylation site in a nuclear localization sequence.In agreement with previous work PP2A-B55 regulated dephosphorylation of several nucleoporins at mitotic exit consistent with Nup153 and TPR being present in the interactomes.These networks will be useful to dissect PP1 and PP2A-B55 regulation in the future.
7. For non-specialists, it may be helpful to define INCENP and explain its role in mitosis.

We have provided a brief description of INCENP function during mitosis for the non-specialist. We write on page 12:
During anaphase, the chromosomal passenger complex (CPC) translocates from chromatin to the central spindle.The CPC is composed of Aurora B, Borealin, Survivin and INCENP and binding to Mklp2 allows translocation of the complex to the central spindle at anaphase hereby facilitating cytokinesis.INCENP T59, a Cdk1 phosphorylation site, must be dephosphorylated to allow binding to Mklp2 and timely translocation (Fig. 3A) [29].
8. Minor question: is "dephosphorylome" a common term?If not, while this is a purely stylistic consideration, it may by simpler for the readers if some more common phrasing is used.
We have used more common phrasing.9.In the last sentence of the first paragraph, there is a typo: "phosphoyraltion" Corrected.

Reviewer #2:
In this study, Hein and co-workers address the complex topic of cellular protein phosphatase selectivity.To do this they use mass spectrometry approaches to analyse dephosphorylation of peptide libraries in vitro by PP1 and PP2A-B55, coupled with global identification of cellular substrates for these enzymes as an in vivo correlate.In summary, there is a considerable amount of high-quality work in the study, and the new data will provide a valuable additional resource for the field.

Specific points:
In Figure 3, INCENP is taken as an example to test a key feature of the dephosphorylation motif.Previous work has shown that INCENP relocalisation from pericentromeric chromatin to the anaphase spindle requires dephosphorylation at T59, and can be prevented with a T59E mutant.This forms a switch controlling binding to a motor protein KIF20A at the onset of anaphase.The charge introduced in the S61E mutant may directly prevent binding to KIF20A.Additional controls are necessary for Figure 3D-E, where a T59A/S61E mutant should be tested.If the authors are correct, then this should to the anaphase spindle since it cannot be phosphorylated.If it fails to localise then defective anaphase localisation is due to a blockade of KIF20A binding and not a dephosphorylation defect.

We now write on page 13:
To determine if the cellular effects of the S61E mutation was purely attributed to a lack of T59 dephosphorylation we investigated if the T59V mutation could rescue the S61E phenotype.However, the INCENP T59V S61E mutant was unable to translocate to the central spindle.This suggests that the S61E mutation both affect T59 dephosphorylation and possibly Mklp2 binding.We note that S61 might constitute a phosphorylation site although it has not been reported in phosphoproteomic screens.Our results argue that INCENP T59 dephosphorylation kinetics are affected by the surrounding sequence and that this could contribute to timely INCENP translocation.
The authors note that while the overlap between PP2A-B55 substrates in vivo in their data and previous work is good, the overlap for PP1 is less clear.There are good reasons for this which need a little more explanation.This also highlights some caveats to the peptide approach, which is important since PP1 is generally thought to act in complex with other regulatory subunits that modify substrate selection.Using PP1 alone in the in vitro approach, and taking Nipp1 as the sole method to modulate PP1 in vivo may not address the full range of PP1 substrates.PP2A-B55 is probably only regulated by Arpp19/ENSA, so the approach would be more general in terms of the range of substrates.
We fully agree with the reviewer on these points.Firstly, we think that previous work using long term RNAi depletion of PP1 are causing too many indirect effects in contrast to our short term inhibition experiments.Secondly previous work adding purified PP1 catalytic subunits to extract might not recapitulate the complexity of PP1 holoenzyme specificity which is why we opted for a strategy to block endogenous PP1 holoenzyme formation through RVxF competition.We think that it is not suprising that these very different approaches would give completely different results.We favor that our strategy in many ways are superior to prior work underscored by the strong number of PP1 regulated sites found in PP1 interactors.However, we phrased our wording more diplomatically in the paper.We have now more carefully explained the differences in the approaches and also pointed out that ARPP19 data is more straightforward to interpret as the active site is inhibited.

We write on page 10:
Our short-term inhibition of PP1 using Nipp1 is quite distinct from a more long-term inhibition using RNAi of PP1 potentially explaining the limited overlap.In contrast asynchronous cell lysate experiments with purified PP1 addition is different from a mitotic lysate where we inhibit RVxF binding and it is not surprising that this will unravel different substrates.As shown later we see a good overlap between PP1 mitotic interactors and PP1 substrates giving us confidence in our ability to identify PP1 regulated sites.

And on page 9:
We used the central domain of Nipp1 and thiophosphorylated Arpp19 (thio-Arpp19) as specific natural inhibitors of PP1 and PP2A-B55, respectively [23][24][25].Nipp1 prevents binding of RVxF-containing proteins to PP1, in this way preventing a large subset of PP1 holoenzymes from forming and thus blocking PP1 dephosphorylation.In contrast, thio-Arpp19 blocks the active site of PP2A-B55, effectively blocking activity.It is important to keep in mind that the inhibitors used here act by distinct mechanisms but a present no specific inhibitors of the PP1 active site exists.
Figure 4 is confusing and it is difficult to extract a clear message.Is there some other way to represent the choice of network diagram is not obvious to me, since the study isn't focussed on the interactions between proteins, but rather the sequences of individual phosphatase sites.Is there some way to strengthen the link to Figure 5 in terms of testing whether dephosphorylation alters behaviour of a key PP1 regulated protein or proteins.In that sense the data in Figure S10 is important and could be moved to a main figure.

We write on page 14:
Our networks revealed that both PP1 and PP2A-B55 dephosphorylate a diverse set of proteins regulating core biological processes involved in mitotic exit as well as interphase functions.Several known RVxF containing proteins such as KNL1, RIF1 and RepoMan (CDCA2) were substrates of PP1 as expected but our network analysis also suggested that these proteins coordinate dephosphorylation of associated proteins.As an example, KNL1 could scaffold PP1 dephosphorylation of CENPC S261, a phosphorylation site in a nuclear localization sequence.In agreement with previous work PP2A-B55 regulated dephosphorylation of several nucleoporins at mitotic exit consistent with Nup153 and TPR being present in the interactomes.These networks will be useful to dissect PP1 and PP2A-B55 regulation in the future.

Reviewer #3:
This manuscript describes an adaptation of the MRBLE peptide:protein interaction assay to a format (MRLBE-Dephos) that can be used to measure time-dependent peptide dephosphorylation by phosphatases.The authors have subsequently used this assay to compare the substrate selectivity of two key phosphatases (PP1 and PP2A-B55), either purified or as present in mitotic cell lysates.The MRLBE-Dephos assay is novel and generated interesting data, but needs to be better validated.Also, while the observed determinants of substrate selectivity of PP1 and PP2A-B55 are largely in agreement with previously established determinants, as derived from other experimental approaches, there are obvious differences that need to be further explored.My detailed comments are detailed below.

Specific comments
1. What are the concentrations of the peptides and phosphatases during the MRLBR-dephos assay?The method section only specifies volumes or amounts.This is essential information needed for for comparison with other phosphatase assays.

To clarify, we have added this information to the Materials and Methods.
2. The MRLBE-Dephos assay measures remaining phosphate (high signal® less high signal), which is less sensitive than classical assays that quantify phosphate release (no signal ® signal).This should be commented upon.

While the MRBLE:dephos assay looks for a decrease in signal, the assay itself includes ~50 replicates per timepoint. As sensitivity is a function of assay modality and statistical power, it's not clear that MRBLE:dephos is necessarily less sensitive than classical assays. We further note that in direct comparisons between MRBLE:dephos assays and classical dephosphorylation assays, MRBLE:dephos appears to discriminate between small differences in dephosphorylation that are not resolved via classical assays (Fig. R2), suggesting that MRBLE:dephos may actually have enhanced sensitivity.
3. In all MRLBE dephosphorylation assays (Fig. S1), the dephosphorylation apparently stops before completion.The reason for this is not explained.

If we examine the raw intensity data over time for each bead code within each experiment, we see 3 main behaviors: 1. Some codes start out with a very high signal intensity that decreases dramatically. This behavior is consistent with a bead that had large amounts of phosphorylated residues at the start of the experiment (time t=0, prior to the addition of phosphatase) that were then removed by the enzyme. Most of these curves for PP1 end up decreasing down to a Cy5 intensity that is within 2-fold of the expected background signal, suggesting that these dephosphorylation reactions have gone to completion. 2. Some codes start out with a very high signal intensity that stays pretty high throughout the course of
the experiment.This behavior is consistent with a bead that had large amounts of phosphorylated residues at the start of the experiment and these phosphorylated residues were unaffected by the addition of the enzyme.In this case, no dephosphorylation takes place because the peptide sequence is not a substrate for the phosphatase and the failure to go to completion is not a concern.

To identify and filter out bead codes with unsuccessful phosphopeptide synthesis, we now impose a requirement that the median intensity for all beads from a given code at the start of the experiment must be greater than a threshold value (calculated as the median + 1 standard deviation of the negative control beads). This requirement eliminates a small number of codes throughout the experiments but ensures that the remaining values represent high quality data. We have edited the Materials and Methods to clarify this.
For the codes that decrease but not all the way to background levels, this behavior is consistent with a population of phosphorylated peptide that is not dephosphorylated by the added phosphatase.

We'd like to again thank the Reviewer for their question -this type of question is a clear instance in which peer review allowed us to reconsider the data and change the data analysis to be significantly more robust.
Also, since the dephosphorylation is not linear with time (Fig. S1), it is not clear how the dephosphorylation score can be accurately derived from the first and last time points.

Beads either are dephosphorylated largely to completion or are not dephosphorylated at all. Thus, the final intensity divided by the starting intensity provides a reasonable metric for the degree of dephosphorylation that occurred over the course of the experiment.
4. A comparison of classical phosphatase assays (phosphate release) and the MRLBE-Dephos score (Fig. 1E) shows essentially no dephosphorylation for some peptides with the first assay but still very significant dephosphorylation with the second assay.This indicates that the MRLBE-Dephos assay not only measures phosphatase activity but, e.g., also loss of bead-associated peptide.

We again appreciate the Reviewer's careful reading and consideration of the data. From looking at the raw data, it's clear that many of the codes do not show any loss of signal over the course of the experiment. This rules out any significant loss of bead-associated peptide over the course of the experiment.
However, the Reviewer is correct that the original figure showed more significant dephosphorylation of some peptides with the second assay.

Going back to the original intensities, it is clear that 1 of the 5 peptides in the MRBLE:dephos assays had low signal intensities at the start of the experiment that did not differ significantly from the negative control intensities, suggesting poor synthesis of phosphorylated peptides on those samples. As described above, we now filter out sequences for which phosphopeptide synthesis was not successful, which eliminates 1 sequence from consideration. We present a revised version of Figure 1F that shows strong correlation between the 2 assays.
5. Since the peptides were synthesized on the beads, it should be explored whether binding to the beads affects their properties as substrates.

While this is a valid concern, it is (unfortunately) beyond the scope of what is possible at this time.
As we are able to measure many peptides in parallel in a single experiment, the most relevant information is the information about how specific amino acid changes change the relative binding affinity.We have indicated these points in the revised manuscript.

We are by no means claiming that MRBLE-Dephos fully represents the complex biological dephosphorylation reactions occurring in cells with complex substrates and holoenzymes, but it provides an approach for high-throughput characterization of dephosphorylation reactions not possible by any other current method.
6.It is biologically not relevant to compare the substrate selectivity of the PP1 monomer, which does not exist in vivo, with a PP2A-B55 holoenzyme.Also, bacterially expressed PP1 has a substrate selectivity that is very different from that of native PP1 (https://doi.org/10.1111/j.1432-1033.1993),making a direct comparison even more irrelevant.

We now write on page 8-9:
In conclusion, we develop a generally applicable high throughput dephosphorylation assay that allows quantitative mapping of protein phosphatase dephosphorylation motifs using only very small amounts of material.We use this to show the existence of optimal dephosphorylation site signatures for PP1 and PP2A-B55, providing a set of rules for accessing whether a phosphorylation site is optimal for one of these phosphatases.In vivo these rules will be further shaped by the PP1 holoenzyme composition as well as substrate interactions.
7. The most striking feature of the comparison between purified PP1 and PP2A-B55 is their similar activity on a broad range of peptide substrates (Fig. 1D; Fig. 2A-D).It is difficult to grasp how the observed minor differences can explain the clearly distinct, albeit somewhat overlapping functions, of these phosphatases in vivo.The differences in substrate specificity would most likely have been much more obvious if a specific PP1 holoenzyme and PP2A holoenzyme were compared, with the free catalytic subunits as reference points (see previous comment).

See our comment above. Indeed, specific holoenzymes of PP1 and binding of PP2A-B55 to specific proteins are major determinants of specificity and we have in our view not ignored this but rather in the second half of the manuscript explored this aspect extensively. However, our data do suggest that there are also inherent preferences in the active site of phosphatases (consistent with previous work) that can contribute.
8. It is not correct to refer to 'benchmark in vitro data with in vivo substrates' (p8, line 1) when the actual comparison is purified phosphatases versus phosphatases present in mitotic lysates.
We have rephrased the sentence.9.While thio-phosphorylated proteins are somewhat resistant to dephosphorylation, they will eventually also be dephosphorylated.Hence, the authors should examine to which extent thiophosphorylated Arpp19 is dephosphorylated in the adopted timeframe(s).Nipp1 is indeed a highly selective inhibitor of PP1 but it only prevents the dephosphorylation of a subset of substrates.This limitation should be indicated.
We are always comparing thio-Arpp19 to Arpp19 S62A ensuring that the regulated sites observed are due to PP2A-B55 inhibition.We have not been able to detect thio-Arpp19 by western blot with our phosphoantibody so we cannot tell whether there is dephosphorylation of thio-Arpp19 occuring during the 5-minute incubation.However, even if thio-Arpp19 was getting dephosphorylated and PP2A-B55 not fully inhibited we are comparing to a sample treated with Arpp19 S62A.We agree that Nipp1 only blocks a subset and have indicated this in the revised manuscript.

We now write on page 9:
We used the central domain of Nipp1 and thiophosphorylated Arpp19 (thio-Arpp19) as specific natural inhibitors of .Nipp1 prevents binding of RVxF-containing proteins to PP1, in this way preventing a large subset of PP1 holoenzymes from forming and thus blocking PP1 dephosphorylation.In contrast, thio-Arpp19 blocks the active site of PP2A-B55, effectively blocking activity.It is important to keep in mind that the inhibitors used here act by distinct mechanisms but a present no specific inhibitors of the PP1 active site exists.As controls, we used Nipp1 with a mutated RVxF motif preventing PP1 binding and Arpp19 S62A that cannot inhibit PP2A-B55.An antibody detecting all prolinedirected threonine phosphorylation (TpP) as well as H3S10 confirmed the specific inhibition of phosphatases upon addition of inhibitors (Fig. S5).We do not know the extent of inhibition but the relative comparison to appropriate controls allows us to identify regulated sites.
10. Less than 10% of the identified mitotic phosphorylation sites (˃ 38,000) were affected by PP1 or PP2A-B55 inhibition.This indicates that the inhibition of these phosphatases was incomplete and/or that it is incorrect to that these phosphatases represent the major mitotic-exit phosphatases.

Both options are a possibility. Since we are only inhibiting for 5 minutes it can also be that certain substrates need longer time to get dephosphorylated. Finally, we cannot exclude residual kinase activity. What is important is the clear difference in regulated sites we observe -we cannot make and are not making any claims on the fact that less than 10% of sites are regulated.
11. p9, first line: H3S29 does not exist.Did the authors aim to refer to H3S28?
The reviewer is correct.We have changed this in the revised manuscript.
12. The limited overlap between PP1 substrates in mitotic lysates and previous substrate-mapping studies is worrysome, and is further evidence that PP1 was only partially blocked in the mitotic lysates.

We now write on page 10:
Our short-term inhibition of PP1 using Nipp1 is quite distinct from a more long-term inhibition using RNAi of PP1 potentially explaining the limited overlap.In contrast asynchronous cell lysate experiments with purified PP1 addition is different from a mitotic lysate where we inhibit RVxF binding, and it is not surprising that this will unravel different substrates.As shown later we see a good overlap between PP1 mitotic interactors and PP1 substrates giving us confidence in our ability to identify PP1 regulated sites.
13.The identified PP1 and B55 interactors (Table S2) are poorly described and not independently validated.Are they regulatory subunits and/or substrates?How much overlap is there with known interactors?

We now write on page 14:
We defined interactomes of PP1g and B55a during mitosis using affinity purification of YFP-tagged subunits and also proximity-dependent ligation using miniTurbo to capture interactions at mitotic exit (Fig. S6) [30,31].This resulted in the identification of 923 PP1 interactors and 141 B55 interactors (Dataset EV2).These identified proteins can constitute both regulators and substrates.
14.The interaction of PP1 with RVxF motifs and its regulation by mitotic phosphorylation of residues within or next to the RVxF motif has already been studied in some detail by various research groups and is not properly referred to in the manuscript.Thank you for sending us your revised manuscript.We have now heard back from the two reviewers who were asked to evaluate your revised study.As you will see below, the reviewers think that the study has improved after the performed revisions.They raise however a series of remaining concerns, which we would ask you to address in a final round of revision.We would also ask you to address some remaining editorial issues listed below.

We have provided additional references for the known phosphorylation dependent regulation of PP1 binding to RVxF motifs.
-Our data editors have noticed some unclear or missing information in the figure legends, please see the attached .docfile.We have also made some minor formatting edits.Please make all requested text changes using the attached file and *keeping the "track changes" mode* so that we can easily access the edits made.
-There are callouts for Table S1-S3, but no such tables are uploaded, please fix these callouts.
-The number of EV Figures is large (typically we allow only 5).We would ask you to provide all these figures in in a PDF called Appendix.Appendix figures should be labeled and called out as: "Appendix Figure S1, Appendix Figure S2... Appendix Table S1..." etc.Each legend should be below the corresponding Figure/ -Source data files need to be reorganized to one file (or zip folder per figure).For Appendix figures, all source data should be provided in a single zip folder.
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As a matter of course, please make sure that you have correctly followed the instructions for authors as given on the submission website.*** PLEASE NOTE *** As part of the EMBO Press transparent editorial process initiative (see our Editorial at https://dx.doi.org/10.1038/msb.2010.72 , Molecular Systems Biology will publish online a Review Process File to accompany accepted manuscripts.When preparing your letter of response, please be aware that in the event of acceptance, your cover letter/point-by-point document will be included as part of this File, which will be available to the scientific community.More information about this initiative is available in our Instructions to Authors.If you have any questions about this initiative, please contact the editorial office (msb@embo.org).----------------------------------------------------------------------------Reviewer #1: Overall, this revised manuscript is substantially improved, and the revisions/responses are extremely thorough.This work will be an important addition to the phosphatase field, both in terms of new methods for in vitro substrate specificity characterization, as well as signaling network information for PP1 and PP2A.
A few minor corrects are suggested below: 1.For Figure 2C, it seems that not all of the C-terminal sequences in the diagram have residue coloring, as the N-terminal ones do.Also, it was a bit confusing to have the +1 P listed as part of the N-terminal sequence, given that it is C-terminal to the pT residue.
2. It's unclear what sets of peptides in 2C this sentence is referring to, "For PP1 N-terminal basic residues could compensate for the negative impact of an acidic or glycine residue in pos +2 (Fig. 2C) ."Is there potentially a typo here?3. Pg. 9: "distinct mechanisms but a present no specific inhibitors of the PP1 active site exists" should be, "but at present..." 4. Pg. 10: "proline-directed threonine phosphorylation (TpP)" should be changed to "pTP" for consistency with the changes in the initial revision.4D, label on the right says "none regulated sites" but should say "non-regulated sites" 6.The network diagram in 4C is no longer blurry, but the one in EV9 is still blurry.

Reviewer #2:
The authors have carried out a thorough revision of the manuscript and responded to most of the questions raised in a clear fashion. in my initial review, I raised a question about the cell biology experiments studying INCENP dephosphorylation, specifically did the mutations preventing B55 recognition also have an effect on the interaction with the kinesin motor needed to carry INCENP in anaphase.This has been addressed in part, but the new data seem to indicate that the B55 mutation does in fact perturb the interaction with the kinesin KIF20A.This means localisation of these INCENP mutants to the anaphase spindle is not possible, irrespective of phosphorylation state.I recommend this imaging data and the accompanying graphs are removed.The biochemical and mass spectrometry analysis is clear and tests the role of B55, however the caveat is the mutant isn't suitable for in vivo functional studies.
Reviewer #3: Reviewer 3 In their reply to comment 1, the authors reported that in their assays the phosphatases concentrations (300 nM for PP2A-B56 and 16,000 nM for PP1) were several orders of magnitude higher than the (peptide) substrates (80 nM in total).This is very worrysome, as enzyme assays need to be performed with substrates in a large molar excess.Even with a turnover number (reactions catalyzed/sec/molecule enzyme) for these phosphatases of only 1 -in reality the turnover number is way higher-, all substrate would be hydrolyzed within 0.5 (PP1) to 20 (PP2A) sec, unless the used phosphatase preparations are largely inactive.Yet, the authors adopted an assay time of 14,400 sec (5h).Moreover, enzyme activities can only be properly derived from the rate of substrate-product conversion up to 30% of substrate depletion because the curve flattens at later time points.It

Reviewer #1:
Overall, this revised manuscript is substantially improved, and the revisions/responses are extremely thorough.This work will be an important addition to the phosphatase field, both in terms of new methods for in vitro substrate specificity characterization, as well as signaling network information for PP1 and PP2A.
A few minor corrects are suggested below: 1.For Figure 2C, it seems that not all of the C-terminal sequences in the diagram have residue coloring, as the N-terminal ones do.Also, it was a bit confusing to have the +1 P listed as part of the N-terminal sequence, given that it is C-terminal to the pT residue.

Our response:
We have corrected this.
2. It's unclear what sets of peptides in 2C this sentence is referring to, "For PP1 Nterminal basic residues could compensate for the negative impact of an acidic or glycine residue in pos +2 (Fig. 2C) ."Is there potentially a typo here?

Our response:
We have corrected the text to clarify that Fig. 2C only shows this for acidic residues.
3. Pg. 9: "distinct mechanisms but a present no specific inhibitors of the PP1 active site exists" should be, "but at present..." Our response: Corrected 4. Pg. 10: "proline-directed threonine phosphorylation (TpP)" should be changed to "pTP" for consistency with the changes in the initial revision.
Our response: Corrected 5. Figure 4D, label on the right says "none regulated sites" but should say "nonregulated sites"

Our response:
14th Oct 2023 2nd Authors' Response to Reviewers Corrected 6.The network diagram in 4C is no longer blurry, but the one in EV9 is still blurry.

Our response:
Corrected in the new appendix file.
Reviewer #2: The authors have carried out a thorough revision of the manuscript and responded to most of the questions raised in a clear fashion. in my initial review, I raised a question about the cell biology experiments studying INCENP dephosphorylation, specifically did the mutations preventing B55 recognition also have an effect on the interaction with the kinesin motor needed to carry INCENP in anaphase.This has been addressed in part, but the new data seem to indicate that the B55 mutation does in fact perturb the interaction with the kinesin KIF20A.This means localisation of these INCENP mutants to the anaphase spindle is not possible, irrespective of phosphorylation state.I recommend this imaging data and the accompanying graphs are removed.The biochemical and mass spectrometry analysis is clear and tests the role of B55, however the caveat is the mutant isn't suitable for in vivo functional studies.

Our response:
We clearly indicated this in the text of the revised manuscript and prefer to keep data as the effect of S61E is two-fold: it affects T59 dephosphorylation but also KIF20A binding.As we clearly state this caveat, we think the data are still relevant to keep.

Reviewer #3:
In their reply to comment 1, the authors reported that in their assays the phosphatases concentrations (300 nM for PP2A-B56 and 16,000 nM for PP1) were several orders of magnitude higher than the (peptide) substrates (80 nM in total).This is very worrysome, as enzyme assays need to be performed with substrates in a large molar excess.Even with a turnover number (reactions catalyzed/sec/molecule enzyme) for these phosphatases of only 1 -in reality the turnover number is way higher-, all substrate would be hydrolyzed within 0.5 (PP1) to 20 (PP2A) sec, unless the used phosphatase preparations are largely inactive.Yet, the authors adopted an assay time of 14,400 sec (5h).Moreover, enzyme activities can only be properly derived from the rate of substrate-product conversion up to 30% of substrate depletion because the curve flattens at later time points.It therefore remains incomprehensible for this reviewer (see comment 3) how phosphatase activities can be derived from the first and last time points of 5h-assays, when all substrate appears to be hydrolyzed at the early time points.In conclusion, the adopted assay does not appear to meet the minimal standards for enzyme assays and does not yield conclusive data.

Our response:
We agree completely with the Reviewer that careful control and quantification of enzyme and substrate concentrations are essential for reporting accurate kinetic parameters and inhibition constants (e.g.k cat , K M , k cat /K M , and K i ).This is typically done by varying substrate concentrations, measuring initial rates during the linear regime of substrate turnover, plotting these rates as a function of substrate concentration, and fitting Michaelis-Menten curves to the data to extract best fit values for these parameters.In the Reviewer's discussion of minimal guidelines and standards for enzyme assays, they appear to be suggesting conditions used to derive these kinetic and thermodynamic constants.We have done these types of analyses extensively in prior work for alkaline phosphatases (see, for example, Markin*, Mokhtari* et al., Science 2021;Markin et al., PNAS 2023).
However, these concerns are not relevant here.We are not attempting to quantify kinetic or thermodynamic constants in MRBLE-dephos assays -and we do not claim to do so.The MRBLE-dephos assay incubates a phosphatase of interest with a selection of bead-bound peptides in a single volume; imaging these beads reveals sequence-specific differences in phosphorylation efficiency over time.We report these relative sequence-specific differences in phosphorylation activity as a "dephosphorylation score" that compares the percentage of remaining phosphates on peptides of each sequence by the end of the assay.This score has no physical meaning (it is not a thermodynamic or kinetic constant); instead, the "dephosphorylation score" simply provides a convenient scalar number that reports on the fact that while some sequences are dephosphorylated efficiently by a given phosphatase, others are unaffected.Peptides with a high "dephosphorylation score" are preferentially desphosphorylated in this in vitro assay relative to other peptides and are therefore also likely to be preferentially dephosphorylated in cells; thus, the "dephosphorylation score" carries useful information about relative sequence preferences.
The authors agree with several other criticisms (points 5, 6, 7, 10, 12), but do not seem to grasp the implications for the interpretation and value of their data.

Our response:
The reviewer pointed to certain limitations of  and the extension of its results to in vivo regulation of phosphatases.Although we have acknowledged these limitations in the revised manuscript, we still find that MRBLE-Dephos provides an important technical advancement and that its results provide important insight into inherent phosphatase specificity.We have been very clear in the manuscript on the role of binding partners in controlling phosphatase specificity.Furthermore, the concerns raised by the reviewer regarding the mitotic lysate data (points 10,12) and approach we do not share.The fact that only 10% of sites are found to be regulated by our approach and for PP1 we see a limited overlap with previous screens was clearly addressed in our previous rebuttal.Molecular Systems Biology charges an article processing charge (APC) to cover the publication costs.You, as the corresponding author for this manuscript, should have already received a quote with the article processing fee separately.Please let us know in case this quote has not been received.
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specificity principles uncovered by MRBLE-Dephos and global substrate identification Dear Jakob,

Fig. R1 .
Fig. R1.Number of beads per code per experiment.Bars and error bars represent the median and standard deviation of the number of beads profiled across all timepoints in each experiment.

Fig. R2 .
Fig. R2.Correlation between results of MRBLE:Dephos and traditional phosphate release experiments.Markers and error bars indicate mean and standard deviation of dephos scores across experiments, black dashed line indicates the identity line, and red dashed line indicates a linear regression.
specificity principles uncovered by MRBLE-Dephos and global substrate identification Dear Jakob, specificity principles uncovered by MRBLE-Dephos and global substrate identification Dear Jakob, Thank you again for sending us your revised manuscript.We are now satisfied with the modifications made and I am pleased to inform you that your paper has been accepted for publication.*** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://dx.doi.org/10.103/msb.2010.72),Molecular Systems Biology publishes online a Review Process File with each accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.If you do NOT want this File to be published, please inform the editorial office at msb@embo.orgwithin 14 days upon receipt of the present letter.LICENSE AND PAYMENT:All articles published in Molecular Systems Biology are fully open access: immediately and freely available to read, download and share.
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